Vaccine against Gram-negative bacterial infections

ABSTRACT

A vaccine, effective in inducing the production of antibodies with which to immunize a second subject passively against infection by Gram-negative bacteria and LPS-mediated pathology, comprises a non-covalent polyvalent complex formed between purified, detoxified LPS derived from  E. coli  and purified outer membrane protein derived from  N. meningitidis . The same vaccine will also actively immunize a host subject against Gram-negative bacterial infections and LPS-mediated pathology. Meningococcal infections are included among those Gram-negative bacterial infections protected against by the vaccine.

This application is a continuation of application Ser. No. 08/230,402,filed Apr. 20, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a vaccine effective against infections withGram-negative bacteria and lipopolysaccharide (“LPS”)—mediated pathologyinduced by Gram-negative bacterial infections. More particularly, itrelates to a non-covalent, polyvalent complex vaccine containingpurified E. coli LPS endotoxin and purified N. meningitidis outermembrane protein, which vaccine produces, in an actively immunizedsubject, an immune response against Gram-negative bacterial infectionand the pathology caused by the LPS endotoxin. The present inventionalso relates to production of specific polyclonal antibodies that can beused to protect a second subject passively against Gram-negativebacterial infections and LPS-mediated pathology.

2. Description of the Background Art

Infections by Gram-negative bacteria and consequent septic shock areleading causes of death among hospitalized patients. It is estimatedthat Gram-negative sepsis has an incidence of 70,000 to 300,000 casesper year in the United States. McCabe et al., Am. J. of Med. 68: 344(1980).

It is well-documented that a principal mediator of Gram-negativebacterial septic shock is a LPS endotoxin present on the outer membraneof Gram-negative bacteria. Luderitz et al., Rev. Infect. Dis. 61: 428(1984); Rietsckel et al., loc. cit. 9 (suppl.): 5527 (1987).

Attempts have been made to produce vaccines that will produceanti-endotoxin antibodies, and thereby protect against septic shock. Fora review, see Cross et al., J. Endotox. Res. 3: 57 (1994). Ziegler et.al., N. Eng. J. Med. 107: 1225 (1982) showed in a clinical setting thatpolyclonal antiserum obtained from volunteers immunized with boiled E.coli J5 (Rc chemotype) provided significant protection. In anotherstudy, however, the human polyclonal antibody to J5 boiled cell vaccinewas not superior to normal human IgG in reducing death fromGram-negative bacteremia. Calandra et al., J. Infect. Dis., 158:312(1988). On the other hand, more recently it was shown thataffinity-purified IgG derived from the serum of rabbits immunized withJ5 boiled cell vaccine afforded neutropenic rats substantial protectionagainst challenge with Ps. aeruginosa, a heterologous Gram-negativebacteria. Bhattacharjee et al., Clin. Res. 41 (2): Abs 247 (1993). Thesecontradictory reports point up the uncertainty and unpredictability ofusing boiled J5 LPS as a vaccine.

Disappointing results also have been reported in the use ofanti-endotoxin monoclonal antibodies. Clinical trials of the HA-1A humanmonoclonal antibody (Ziegler et al., N. Eng. J. Med. 324: 429 (1991)),and the E5 murine monoclonal IgM antibody (Greenman et al., J. Am. Med.Assoc. 266: 1097 (1991); Wenzel et al., 31 st Intl. Conf. Antimicrob.Agts. Chemotherapy 240, Abstr. 1170 (1991)) did not generate dataadequate to support product licensing. Cross et al. (1994), above.

Earlier, Kanegasaki et al. in BACTERIAL ENDOTOXIN: CHEMICAL, BIOLOGICALAND CLINICAL ASPECTS, Homma et al., eds. (Verlag Chemie, 1984),complexed various crude LPS preparations with an outer membrane proteinderived from E. coli and compared these complexes in two systems,namely, induction of interferon production in rabbit spleen cells andactivation of preclotting enzymes of the horseshoe crab. It was reportedthat differing degrees of activity were exhibited by complexes derivedfrom different LPS preparations, and that substituents not masked aftercomplex formation are in part responsible for the variability ofactivity. This, in turn, may be a reflection of the great variability ofthe O-polysaccharide chain structure of LPS's even among differentstrains of the same species. For example, there are over 100 serotypesof E. coli based on the structure of O-polysaccharide. Kenne et al. in 2POLYSACCHARIDES 282, G. O. Asoinall, eds. (1983).

Because of these uncertainties and an unmet need of long-standing for avaccine effective against Gram-negative bacterial infections, thepresent inventors have devised a novel vaccine which allows for bothactive and passive immunization against Gram-negative bacterialinfections.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a means ofboth actively and passively immunizing a subject against Gram-negativebacterial infections and LPS pathology. In this regard, a subject can beactively immunized with a non-covalent vaccine comprising a complexbetween purified E. coli LPS and purified outer membrane protein (“OMP”)derived from N. meningitidis. Serum or plasma from an actively immunizedsubject, or IgG isolated therefrom (hereafter “specific polyclonalantibody”), can be administered to a second subject to confer on thelatter a passive protection against Gram-negative bacterial infectionsand LPS-mediated pathology, including sepsis.

It is another object of the present invention to provide a purified,detoxified LPS endotoxin from an E. coli strain suitable for use in theaforementioned non-covalent complex vaccine.

It is still another object of this invention to provide a purified outermembrane protein from an N. meningitidis strain suitable for use in theaforementioned non-covalent complex vaccine.

It is yet another object of the invention to provide a non-covalentcomplex between purified, detoxified LPS endotoxin from E. coli andpurified outer membrane protein from N. meningitidis.

Another object of the present invention is to provide a method ofpassively immunizing a subject against Gram-negative bacterialinfections using plasma or post-immune serum (antiserum), or specificpolyclonal antibody purified therefrom, obtained from a host subjectactively immunized with the aforementioned non-covalent complex vaccine.

Yet another object of the invention to provide a method of using thenon-covalent complex vaccine of the invention for active or passiveimmunization of a subject against meningococcal infections.

These and other objects will become apparent by reference to thespecification and examples below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows survival data from the neutropenic rat model of sepsis,wherein the rat is treated with IgG isolated from the post-immune serumof a rabbit immunized with J5 LPS-GBOMP non-covalent complex vaccine (R#62, post-immune serum IgG, +--+); IgG isolated from the serum of rabbit# 42374 that was immunized against J5 DLPS-GBOMP (Δ--Δ); Preimmunerabbit serum IgG (-o-); PBS control (+--+).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, a non-covalent, polyvalent complexbetween purified, detoxified LPS (“DLPS”) derived from E. coli andpurified outer membrane protein derived from N. meningitidis is providedwhich, when injected into a host subject, actively immunizes the hostsubject against Gram-negative bacteria and LPS-mediated pathology.Post-immune serum or plasma from the host subject, or specificpolyclonal antibody purified from these fluids, can be administered to asecond subject, passively immunizing the second subject againstinfection by Gram-negative bacteria and LPS-induced pathology.

The preferred strain of E. coli from which to prepare purified anddetoxified LPS endotoxin is an E. coli J5 (Rc chemotype) strain. NativeJ5 LPS may be purchased from List Biological Labs, Inc., Campbell,Calif. For purification purposes it is preferred that the LPSpreparation contain less than about 1% protein and less than about 1%nucleic acid. By “purified E. coli LPS” is meant LPS suitable for use inthe invention vaccine prepared by sonicating native LPS in an alkalinesolution, heating the solution at 65° C., neutralizing the cooledsolution to pH 7.0, removing released fatty acids and remaining nativeLPS by Sephadex G-50 chromatography, and collecting the purified,detoxified DLPS. As determined in a standard rabbit pyrogenicity test,this method reduces the pyrogenicity of LPS preparations, and thus arealso referred to as DLPS. An embodiment of this procedure is describedin Example 1 below.

A preferred strain of meningococcus is N. meningitidis group B. Theouter membrane protein therefrom (hereinafter “GBOMP”) is prepared asdescribed in Zollinger et al., J. Clin. Invest. 63:836 (1079), and U.S.Pat. No. 4,707,543 (1987), the contents of which are incorporated hereinby reference. Briefly, meningococcal group B cells are warmed to about55 to 60° C. for a brief period, and disrupted in a shearing device suchas OMNIMIX, sold by DuPont Instruments (Newtown, Conn.). The shearate iscentrifuged at forces up to 100,000×g to isolate in the pellet the outermembrane complex (“OMC”). The OMC is dissolved in buffered detergent,and repeatedly fractionated with ammonium sulfate so as to collectpurified GBOMP precipitating at 0.5 g/ml salt. The protein is thenultrafiltered through a membrane. This preparation will be referred toherein as “purified outer membrane protein”. Details of one embodimentare provided in Example 2 below.

To prepare the inventive non-covalent complex, solutions of GBOMP and J5LPS are mixed, incubated at room temperature until complex formation hasoccurred (0.5 to 2 hrs.), and dialyzed repeatedly against sterileisotonic saline for 3 to 5 days in the cold. Any insoluble material isremoved by centrifugation and by filtering through a membrane (e.g.,0.45 μm, Amicon). The vaccine complex is preferably stored in the cold(5° C.) until use. The protein concentration of the vaccine is generallyadjusted to about 1 to 3 mg/ml. In general, the ratio of GBOMP to J5 LPSin such complexes is about 1–2. Embodiments of this procedure areprovided in Examples 3 and 4.

The immunogenicity of the J5 LPS-GBOMP non-covalent complex vaccine maybe tested in a rabbit system. Rabbits (preferably New Zealand whiterabbits, Hazelton Res. Prods., Denver Pa.) are injected intramuscularlywith a sterile saline solution of the above-described complex vaccine.It is preferred that each rabbit receive a total of 3 doses of thevaccine. Control animals may be used to test variables, such asindividual components of the vaccine complex and vaccine dosage.Post-immune serum is collected from the immunized rabbits, and theamount of anti-LPS antibody present in their serum determined by anELISA test. IgG can be isolated from this post-immune serum or plasmaconventionally. Two embodiments of this procedure are shown in Example 7and Example 8, respectively.

The aforementioned ELISA test is performed in microtitre platesessentially by the method of Engvall et al., J. Immunol. 109: 129(1972), with slight modifications as will be described in detail inExample 6 below. Briefly, wells are coated with poly-L-lysine (SigmaChem. Co., St. Louis, Mo.), and coats overlaid with either J5 LPS orlipid A. Antigen (Ag)-coated plates, after blocking nonspecific bindingsites with a foreign protein, for example, casein, are incubated withserial dilutions of the rabbit serum containing antibodies (Ab) Asecond, enzyme-tagged antibody (Ab-E) is added to form an Ag-Ab₁–Ab₂-Ecomplex, and the presence and amount of Ag determined calorimetricallywith a chromogen (p-nitrophenyl phosphate). Absorbancies may bedetermined automatically, for example, using the DYNATECH PLATE READERproduced by Dynatech (Alexandria, Va.). ELISA absorbancy units arecalculated by multiplying the serum dilution by the A_(410nm) at anabsorbancy reading near the midpoint of the linear portion of thestandard curve. These procedures are described in Example 6 below.

A vaccine within the present invention will be useful for activeimmunization of populations at risk of acquiring septic shock, such assurgery patients, the military, police and firemen. In addition, humanvolunteers can be safely actively immunized with this non-covalentcomplex, and antibodies prepared from such human hyperimmune sera can beused for passive protection of patients, including domestic and otheranimals, against Gram-negative bacterial infections and sepsis.

The above-described non-covalent complex is simple to prepare and ishighly cost effective. Unlike the original boiled J5 LPS vaccineemployed by Ziegler et al. (1982) above, the present complex is preparedfrom purified, detoxified J5 LPS and purified GBOMP, and is thus welldefined and preferable over the prior art boiled whole bacterial cellvaccines. The J5 LPS can be prepared in large quantities suitable forclinical use.

This is the first successful use of a purified, detoxified J5 LPS in avaccine formulation.

The following examples are intended to illustrate preferred embodimentsof this invention, and are not intended to limit the scope of theinvention which is defined by the specification and appended claims.

EXAMPLE 1 Preparation of Purified, Detoxified J5 LPS (J5 DLPS)

The lipopolysaccharide (LPS) from E. coli J5 (Rc chemotype, J5 LPS, lot#16A) was purchased from List Biological Laboratories Inc. (Campbell,Calif.). This preparation contained less than 1% protein and less than1% nucleic acid as determined by absorbances at 260/280 nm.

In view of the fact that the native J5 LPS was pyrogenic in the rabbitpyrogenicity test at a dose of 0.01 μg, it was necessary to prepare adetoxified J5 LPS for use in making a J5 DLPS-GBOMP non-covalent complexvaccine.

Native E. coli J5 LPS (10 mg) was dissolved into 4.5 ml of 0.1 M NaOHsolution, and then sonicated for 5 minutes. The slightly hazy solutionwas heated in a screw-capped tube at 65° C. for 2 hours. The cooledsolution was neutralized with 1.0 M HCl to a pH of about 7.0. Thereleased fatty acids and any remaining native J5 LPS were removed bychromatography on Sephadex G-50 (1.6×60 cm) using 0.01 Mpyridine-acetate buffer pH 6.5 as eluant. The purified, detoxified J5LPS (J5 DLPS) eluted shortly after the void volume. The fractions werecombined and lyophilized (yield=6.5 mg). Such preparations werepyrogen-free at the 0.5 μg level of DLPS (see Example 5).

EXAMPLE 2 Purification of N. Meningitidis Group B Outer Membrane Protein

Neisseria meningitidis GBOMP was prepared by methods describedpreviously. See Zollinger et al., J. Clin. Invest. 63: 836–48 (1979),and U.S. Pat. No. 4,707,543, the respective contents of which areincorporated herein by reference. Briefly, meningococcal group B cellsfrom strain #8529 (collection of Walter Reed Army Institute of Research,Washington, D.C.) from a 15 liter culture, collected by continuouscentrifugation (135 g, wet wt.), were suspended in 300 ml buffercontaining 0.05 M Tris-chloride, 0.15 M NaCl and 0.01 M EDTA, pH 7.4 andwarmed at 56° C. for 30 minutes. The suspension, cooled to roomtemperature, was sheared in an Omnimixer (DuPont Instruments, Newton,Conn.) at full speed for 3 minutes and centrifuged at 30,000×g for 20minutes. The pellets were re-extracted in the same way and thesupernates were combined. The combined supernate was centrifuged at25,000×g for 15 minutes. The resulting supernate was centrifuged at100,000×g for 1 hour, and the pelleted outer membrane complex (OMC) wassuspended in about 150 ml of distilled water by magnetic stirring. Thesuspension was centrifuged at 10,000×g for 15 minutes and the resultingsupernate was centrifuged at 100,000×g for 1 hour. The pelleted OMC wassuspended in about 75 ml of distilled water and to this suspension wasadded 75 ml of 2× TEEN buffer (2% Empigen BB, 0.35 M NaCl, 0.021 M EDTA,0.10 M Tris-HCl, pH 8.0). The mixture was magnetically stirred for 1 h.

Solid ammonium sulfate (500 g/L) was added to the OMC suspension, andthe mixture was stirred until all the ammonium sulfate was dissolved.The suspension was allowed to stand at room temperature for 1 hour andthen centrifuged at 20,000×g for 20 minutes. The precipitated proteincollected at the top of the tube was recovered by drawing off liquidfrom the bottom. The protein was redissolved in 150 ml of TEEN buffer(1% Empigen BB, 0.15 M NaCl, 0.01 M EDTA and 0.05 M Tris-HCL, pH 8.0).The precipitation was repeated two more times using 600 g/L of ammoniumsulfate. The final precipitate was dissolved in TEEN buffer at 1–2 mg/mland dialyzed against 4 changes of 20 volumes of TEEN buffer (containing0.1% Empigen BB) to remove the ammonium sulfate. This outer membranepreparation was combined with more OMP extracted from the pelleted cellsusing detergent as follows.

The pelleted cells were suspended in about an equal volume of 1 M sodiumacetate buffer pH 4.9 and 3 volumes of distilled water were added. Tothis mixture was added 5 volumes of a solution containing 6% Empigen BBin 1.0 M calcium chloride. The mixture was stirred at room temperaturefor 1 h., after which ethanol was added to a concentration of 20%volume/volume. The resulting precipitate was removed by centrifugationat about 20,000×g for 10 minutes. The pellets were discarded and thesupernatant was brought to 45% ethanol volume/volume. The precipitatedproteins were collected by centrifugation at about 20,000×g for 10minutes and dissolved in TEEN buffer. Any insoluble material was removedby centrifugation at about 20,000×g for 10 minutes. The protein wasfurther purified to remove lipopolysaccharides, capsular polysaccharideand nucleic acid by ammonium sulfate precipitation three times asdescribed above.

The GBOMP prepared by the two sequential methods were combined andconcentrated by ultrafiltration on a PM-10 membrane. The final proteinconcentration was 3.67 mg/ml.

EXAMPLE 3 Preparation of J5 DLPS-NMGBOMP Non-Covalent Complex

N. meningitidis group B OMP (NMGBOMP) solution 1.5 ml (3.67 mg/ml,Example 2) was added to 5.0 ml of a solution (0.8 mg/ml) of J5 (DLPS in0.9% NaCl. The mixture was kept for 1 hour a room temperature and wasthen dialyzed against 100 volumes of sterile 0.9% NaCl at 5° C. for 48hours. The dialysis buffer was changed and dialysis continued at 5° C.for another 72 hours. The dialyzed solution (5.5 ml) was slightly hazy.This solution was filtered through 0.45 μm membrane and stored at 5° C.Analysis showed that this J5 DLPS-NMGBOMP non-covalent complex vaccinehad J5 DLPS=600 μg/ml and NMGBOMP=1.0 mg/ml.

EXAMPLE 4 Preparation of J5 LPS-N. Menigitidis GBOMP Non-covalentComplex

In another preparation, 5 mg of J5 LPS was dissolved in 5 ml of sterile0.9% NaCl for injection, USP (Kendall and McGaw, lot #JOBO29A). Thisgave a hazy suspension. The suspension was sonicated for 10 minutes inan Ultrasonic bath (Branson, model 5200). It remained a hazy suspension.1.4 ml of GBOMP solution (3.67 mg/ml from Example 2) was added to the J5LPS suspension. The mixture became clear immediately. This clearsolution was dialyzed in 150 volumes of sterile 0.9% NaCl (injectionquality, USP) at 5° C. for 5 days. The dialyzed solution was slightlyhazy. The insoluble material was removed by centrifugation at 10,000×gfor 20 minutes. The clear supernate (vaccine #1) was stored at 5° C.until used. The ratio of GBMOMP:J5 LPS was found to be 1.5:1 (w/w). Aportion (3.0 ml) of this preparation was filtered through a 0.45 μmmembrane. The filtered sample (vaccine #2) was stored at 5° C. untilused. This second vaccine addresses the possible effect of a filtrationstep in the preparation of a sterile vaccine. The ratios are slightlyaltered. The mass ratio of GBOMP to J5 LPS in representative complexeswas 1.2:1.

EXAMPLE 5 Test for Pyrogenicity of the J5 DLPS-NMGBOMP Complex

The J5 DLPS-NMGBOMP non-covalent complex vaccine formulation was testedfor pyrogenicity by the standard rabbit pyrogenicity assay. This vaccinewas not pyrogenic at a dose containing 0.5 μg J5 DLPS. At a 10-foldhigher dose (5.0 μg J5 DLPS) it was pyrogenic with an average rise intemperature of 1.3° C. (see Table 1). Based on these results, a dosecontaining 1.0 μg of J5 DLPS is selected. Converting this to a humandose for a 70 kg volunteer, a dose of about 35 μg of J5 DLPS is selectedto provide high immunogenicity in humans.

TABLE 1 MAXIMAL Lot # SAMPLE DOSE μg RABBIT # RISE ° C. AVERAGEPyrogenicity AKBXV80.1 J5 LPS-OMP 0.06 2789 1.2 AKBXV80.1 J5LPS-OMP 0.062790 1.6 AKBXV80.1 J5LPS-OMP 0.06 2791 1.2 1.3 AKBXV80.2 J5 dLPS-OMP0.05 2780 0.1 AKBXV80.2 J5 dLPS-OMP 0.05 2781 0.4 AKBXV80.2 J5 dLPS-OMP0.05 2782 0.1 0.2 PASS AKBXV80.2C J5 dLPS-OMP 0.5 2783 0.5 AKBXV80.2C J5dLPS-OMP 0.5 2784 0.0 AKBXV80.2C J5 dLPS-OMP 0.5 2785 0.1 0.2 PASSAKBXV80.2D J5 dLPS-OMP 5.0 2788 1.6 AKBXV80.2D J5 dLPS-OMP 5.0 2801 1.3AKBXV80.2D J5 dLPS-OMP 5.0 2802 1.4 1.4 AKBXV80.4B J5 dLPS 0.05 2792 1.1AKBXV80.4B J5 dLPS 0.05 2793 1.1 AKBXV80.4B J5 dLPS 0.05 2794 1.7 1.3AKBXV80.4C J5 dLPS 0.5 2779 2.1 AKBXV80.4C J5 dLPS 0.5 2786 2.3AKBXV80.4C J5 dLPS 0.5 2787 2.0 2.1

EXAMPLE 6 Enzyme-linked Immunosorbent Assay (ELISA)

The ELISA was performed in 96 well flat-bottom polystyrene microtiterplates (Costar, Cambridge, Mass.) essentially by the method of Engvallet al. above) with slight modification. The wells were first coated with50 μg/ml poly-L-lysine type VIIB in PBS (0.01 M Na-phosphate, 0.14 MNaCl, 0.02% NaN₃ pH 7.4) 100 μl, at 37° C. for 1 hour. The wells wereemptied and then overlaid with either J5 LPS or lipid A at 10 μg/ml inPBS for 3 hours at 37° C. Excess binding sites were then blocked with 1%casein (Fisher Scientific Co., Columbia, Md.) in PBS at 37° C. for 1hour. The wells were washed with PBS between steps to remove unboundmaterial. The antigen-coated plates were incubated with serial 2-folddilutions of antibodies for 16 hours at room temperature (25° C.).Incubation with the second antibody was performed for 20 hours at roomtemperature. Disodium p-nitrophenylphosphate (Sigma Chemical Co.) at aconcentration of 1 mg/ml in (1.0 M diethanolamine buffer, with 1 mMMgCl₂), pH 9.8 was used as the substrate. Absorbance was read on aDynatech plate reader (Dynatech; Alexandria, Va.) at 410 nm. The ELISAO.D. units were calculated by multiplying the dilution of the serum withthe absorbance at 410 nm at an O.D. reading near 0.5. The O.D. readingof 0.5 is at about the midpoint of the linear part of the O.D. vsdilution curve in our assay.

EXAMPLE 7 Immunogenicity of J5 LPS-N. Meningitidis Non-covalent ComplexVaccine in Rabbits

Two groups of 2 each New Zealand white rabbits (Hazelton ResearchProducts, Denver, Pa.), were immunized with the two vaccines in salineby intramuscular injection. The immunogenicity data are shown in Table2. Each rabbit received a dose containing 50 μg of GBOMP. Rabbits #62and 63 each received vaccine #1 which has 50 μg GBOMP and 33 μg J5 LPSin the complex. Rabbits #64 and 65 received the vaccine that wasfiltered through a 0.45μ membrane (vaccine #2); 50 μg GBOMP and 41 μg J5LPS were present in each dose. The rabbits received a total of threedoses of vaccine. As shown in Table 2, the rabbits showed about a 40 to142-fold rise in ELISA titer against J5 LPS four weeks after the firstinjection and another 4 to 6-fold rise one week after the secondinjection. The ELISA titers dropped somewhat after the second injection,but rose after the third injection. The ELISA titers against lipid Awere much lower, and showed only a marginal rise of 1.2 to 2.5-fold overpre-immunization levels.

TABLE 2 Immunogenicity of J5 LPS-N. meningitidis GBOMP Non-covalentComplex Vaccine in Rabbits ELISA Titers of Pre- and Postbleed Sera FromRabbit # 62–65* Rabbit# Prebleed 2/18/92 2/25/92 3/18/92 4/07/92 FOLDElisa Titer in O.D. Units vs J5 LPS 62 106 3,955 25,804 7,014 20,019 18863 99 4,115 14,873 7,411  8,332  84 64 32 3,558 NA 3,142  6,054 189 6532 4,550 16,384 3,891 13,900 434 ELISA Titers vs E. coli Lipid A 62 93  147   276   163   236 2.5 63 185   281   323   281   261 1.4 64 68  97 NA   83   124 1.8 65 270   341   364   334   345 1.2 The firstinjection was given on 1/16/92; the second injection was given on2/18/92 and the third injection was given on 3/18/92. *Rabbit #62–63received the vaccine #1 and rabbit #64–65 received the vaccine #2. NANot available because serum was lost due to breakage of tube.

EXAMPLE 8 Immunogenicity of J5 DLPS-NMGBOMP Vaccine in Rabbits

Five groups of New Zealand white rabbits (2 rabbits in each group) wereimmunized with the J5 DLPS-NMGBOMP non-covalent complex vaccine. Group#1 received the complex containing 25 μg J5 DLPS. Group #2 received thesame dose+QS21 (a saponin adjuvant). Group #3 received the complexcontaining 2 μg J5 DLPS. Group #4 received the same dose as group#3+QS21. Group #5 was a control group receiving 25 μg J5 DLPS (withoutNMGBOMP)+QS21. All rabbits were given 3 doses of vaccine at intervals oftwo weeks. The immunogenicity data are shown in Table 3. The post-immunesera from rabbits in groups 1–4 showed a 30 to 1600-fold rise in titeragainst the J5 LPS. There was no significant difference between group #1and 2. The ELISA antibody titers against NMGBOMP showed 100 to 300-foldrise in titer. Again there was no significant difference between groups#1 and 2, indicating that the QS21 did not enhance the immune responseto the DLPS component of this vaccine.

TABLE 3 ELISA Titers of Pre- and Post-Bleed Sera from Rabbits Immunizedwith J5 DLPS-GBOMP Non-covalent Complex Vaccine Group Rabbit # PrebleedPost-1 Post-2 Post-3 ELISA Titers in O.D. Units vs J5 LPS 1 44660 961,980 3,987 3,430 42374 52 2,035 5,299 8,243 2 44760 151 1,139 3,2193,961 44877 206 1,856 2,816 2,688 3 46170 32 226 1,891 1,392 46880 40432 3,513 4,614 4 40004 33 345 7,052 3,622 46298 305 635 3,276 4,012 546277 104 90 106 124 46886 24 31 37 51 ELISA Titers in O.D. Units vs PA134VA LPS 1 44660 59 296 150 144 42374 1,092 1,067 1,238 3,347 2 447601,187 2,297 1,388 1,907 44877 75 78 88 78 3 46170 10 16 22 22 46880 183652 753 753 4 40004 148 209 198 154 46298 30 43 52 98 5 46277 13 23 3834 46886 93 312 432 355 ELISA Titers in O.D. Units vs. N. meningitidisGBOMP 1 44660 141 851 13,337 12,070 42374 79 580 14,684 26,137 2 44760434 3,072 20,940 25,472 44877 109 1,148 17,792 17,958 3 46170 182 1851,811 2,588 46880 119 296 3,859 4,294 4 40004 116 325 9,497 13,145 46298225 398 5,516 7,577 5 46277 72 81 109 119 46886 84 316 371 377 GR #1Received JS DLPS-GBOMP Vaccine 25 μg GR #2 Received J5 DLPS-GBOMP + QS21Vaccine 25 μg GR #3 Received JS DLPS-GBOMP Vaccine 2.0 μg GR #4 ReceivedJ5 DLPS-GBOMP + QS21 Vaccine 2.0 μg GR #5 Received J5 DLPS + QS21Vaccine 25 μg VACCINE:QS21 RATIO = 1:2 Post-1 = 2 weeks post primaryimmunization Post-2 = 2 weeks post secondary immunization Post-3 = 2weeks post tertiary immunization

EXAMPLE 9 Bactericidal Antibody Response

The post-immune sera from the rabbits of Example 8 was bacteriocidalagainst both homologous (#8529) and heterologous strains (#44/76, #8566and #8047) of Group B meningococcus. As shown in Table 4, this vaccineelicited significant increases (4 to 32-fold) in bactericidal titeragainst both homologous and heterologous strains. Therefore, protectionby this vaccine against group B meningococcal infection will befurthered by the antibodies to group B meningococcal outer membraneprotein induced by the vaccine.

TABLE 4 Bactericidal Titers of Rabbit Sera Group Rabbit # PrebleedPost-1 Post-2 Post-3 Fold Bactericidal Titers vs N. menigitidis Gr. B#8529 1 44660 4 8 32 32 8 42374 8 8 32 16 4 2 44760 8 16 128 128 16 44877 16 16 64 64 4 3 46170 16 16 16 16 — 46880 16 16 16 16 — 4 40004 1616 32 64 4 46298 8 16 16 64 8 Bactericidal Titers vs N. menigitidis Gr.B #8047 1 44660 8 8 8 8 — 42374 4 8 8 8 2 2 44760 4 8 8 16 4 44877 16 1616 16 — 3 46170 16 16 16 16 — 46880 8 8 8 8 — 4 40004 4 16 16 16 4 4629816 16 16 16 — Bactericidal Titers vs N. menigitidis Gr. B #8566 1 4466032 32 64 64 2 42374 32 32 64 32 2 2 44760 16 32 128 128 8 44877 32 32128 64 4 3 46170 64 64 64 64 — 46880 32 32 32 32 — 4 40004 32 32 64 64 246298 32 64 64 128 4 Bactericidal Titers vs. N. menigitidis Gr. B #44/761 44660 4 4 32 32 8 42374 8 8 32 8 4 2 44760 4 16 128 128 32  44877 8 864 64 8 3 46170 8 16 16 16 2 46880 4 4 16 8 4 4 40004 8 8 32 64 8 462988 32 32 64 8 Strain #8529 = B:15:P1.3:L3, 7, 9 Strain #8047 =B:2b:P1.2:L2, 4 Strain #8566 = B:4:P1.15:L3, 7, 9 Strain #44/76 =B:15:P1.16:L3, 7, 9

EXAMPLE 10 Cross-reactivity of IgG from Post-Immune Serum of Rabbit#42374

IgG was isolated from the post-immune serum of rabbit #42374 (J5DLPS-NMGBOMP vaccine) by affinity chromatography on Protein G-Sepharose.IgG was also isolated from pre-immune rabbit serum as a control.Bacteria grown, washed, and incubated with normal or J5 vaccine IgG,followed by FITC-labeled goat anti-rabbit IgG, and then evaluated byFACS analysis showed that IgG from the post-immune serum hadsignificantly higher binding to a broad spectrum of gram-negativebacteria (see Table 5) than the IgG prepared from the pre-immune serum.For bacteria culture conditions in these examples, see Bhattacharjee etal., J. Infect. Dis., (1994), the contents of which are incorporated byreference.

In the absence of treatment with antibiotic (Imipenem) to expose theendotoxin core, the J5 specific IgG showed enhanced binding to at least5 of the bacteria. With antibiotic treatment, there was enhanced bindingof J5 specific IgG to all Gram-negative bacteria. There was no enhancedbinding of the J5 specific IgG to the Gram-positive organism, S. aureus,which lacks endotoxin.

TABLE 5 Binding of Detoxified J5/Group B Meningococcal Outer MembraneProtein Vaccine-induced IgG to Imipenem- treated and Untreated WholeBacteria Normal J5-specific No Rabbit IgG No Rabbit IgG Strain ImipenemImipenem Imipenem Imipenem E. coli 35 0.38 37.6 11.5 40.6 S. aureus 10.66.7 6.8 1.4 E. coli 2961 22.3 20.1 35.9 57.8 PA 2967 0.4 3.4 1.0 35.0 E.cloacae 0.4 2.2 0.9 55.0 2960 0.7 0.0 2.4 18.4 3037 0.4 0.0 1.1 22.9 PA2094 30.0 24.0 78.9 68.9 EC2186 19.4 16.1 22.3 52.3 E. aerogenes 5.7 3.411.8 41.7 E. cloacae 2203 52.1 55.0 49.0 86.7 KP 2085 23.3 37.4 65.995.3

Data are expressed as percent population positive by FCAS. Positive gatedefined by line drawn defining <5% non-specific binding (i.e., additionof secondary antibody, FITC-anti-rabbit IgG, to bacteria in absence ofanti-rabbit IgG).

Bacteria grown, washed, treated with imipenem or buffer, washed,incubated with normal or J5 vaccine-induced rabbit IgG, followed byFITC-labelled goat or anti-rabbit IgG. At least 5,000 bacterialparticles evaluated.

EXAMPLE 11 Protection of Rats in the Neutropenic Rat Model of Sepsis

The neutropenic rat model has been described before. See Collins et al.,J. Infect. Dis. 159: 1073 (1989); Opal et al., J. Infect. Dis. 161: 1148(1990). Briefly, female Sprague-Dawley rats (125–175 g) were obtainedfrom Charles River Breeding Laboratories (Wilmington, Mass.) Cefamandolewas given intramuscularly at a dose of 100 mg/kg beginning 96 hoursbefore bacterial challenge. Cyclophosphamide was given intraperitoneallyat a dose of 150 mg/kg at time 0 and at a dose of 50 mg/kg at 72 hours.At times 0, 48 and 96 hours the challenge strain of P. aeruginosa wasgiven orally via an orogastric tube. Animals were monitored for feverwith a Horiba non-contact digital infrared thermometer (Markson Science,Phoenix) and received antiserum or IgG fractions derived from theantiserum, at 9.0 ml/kg, intravenously via tail vein at the onset offever (temperature >38.0° C., which was usually day 5 or 6). Controlanimals received normal saline on the same schedule. The animals wereobserved for 12 days and deaths were recorded. In a preliminaryexperiment, purified IgG was given at 3.0 ml/kg, 6.0 ml/kg and 9.0 ml/kgrespectively (Total IgG=1.6 mg/ml) to three groups of rats. Bloodsamples were collected from the rats at 1, 6 and 24 hours post infusionand were analyzed by ELISA for anti-J5 LPS titers. The rat sera werealso analyzed for endotoxin content. In this model neutrophils typicallybegin to return on days 9 to 10, and this is monitored by blood smearanalysis on approximately 10% of animals during each experiment.

IgG was isolated from the post-immune serum of rabbit #62 which had beenimmunized with J5 LPS-NMGBOMP non-covalent complex vaccine. This IgGgave significant protection against challenge with lethal doses of avirulent strain of P. aeruginosa 12:4:4. Six out of 10 rats (60%) wereprotected compared to none of 10 rats treated with the controlpre-immune serum IgG (p<0.02) see FIG. 1.

IgG was also prepared from the preimmune serum of rabbit #42374 whichhad been immunized with the J5 DLPS-NMGBOMP complex vaccine. Five of tenrats were protected by passive transfer of this IgG compared to none often rats treated with the preimmune serum IgG (P<0.02); see FIG. 1.

1. A vaccine comprising a non-covalent complex of a purified, detoxifiedlipopolysaccharide endotoxin of Escherichia coli strain J5 and apurified outer membrane protein of Neisseria meningitidis, wherein saidvaccine is effective in immunizing a subject against infection byheterologous Gram negative bacteria by inducing IgG specific toEscherichia coli J5 LPS wherein said IgG binds to, or is cross-reactivewith heterologous Gram negative bacteria.
 2. The vaccine of claim 1,wherein said Escherichia coli strain J5 is of the Rc chemotype.
 3. Thevaccine of claim 1, wherein said Neisseria meningitidis is group BNeisseria meningitidis.
 4. The vaccine of claim 1, wherein the weightratio of said purified outer membrane protein to said purifieddetoxified endotoxin in said non-covalent complex is between 1 and
 2. 5.The vaccine of claim 1, 2 or 3 comprising sterile 0.9% sodium chlorideor QS21 adjuvant.
 6. A method of actively immunizing a subject againstinfection by heterologous Gram negative bacteria comprisingadministering to said subject an immunizing dose of the vaccine ofclaim
 1. 7. The method of claim 6, wherein said Escherichia coli strainJ5 is of the Rc chemotype.
 8. The method of claim 6, wherein saidNeisseria meningitidis is group B Neisseria meningitidis.